Epoxide hydrolases (EH, E.C.3.3.2.3) are enzymes which catalyze the hydrolysis of epoxides including arene oxides to their corresponding diols by the addition of water. EHs play an important role in the metabolism of a variety of compounds including hormones, fatty acid derivatives, chemotherapeutic drugs, carcinogens, environmental pollutants, mycotoxins, and other harmful foreign compounds.
Several members of this ubiquitous enzyme sub-family have been described based on substrate specificity and subcellular localization. Mammalian EHs include cholesterol epoxide hydrolase, leukotriene A.sub.4, hydrolase, hepoxilin hydrolase, microsomal epoxide hydrolase (mEH), and soluble epoxide hydrolase (sEH). The latter two enzymes have been extensively studied and found to have broad and complementary substrate selectivity. The microsomal and soluble forms are known to detoxify mutagenic, toxic, and carcinogenic xenobiotic epoxides, are involved in physiological homeostasis, and both are members of the .alpha./.beta.-hydrolase fold family.
U.S. Pat. No. 5,445,956, issued Aug. 29, 1995, inventors Hammock et al., discloses recombinant human and mouse soluble epoxide hydrolase. The mouse enzyme provides a rodent model to evaluate for therapeutic development of human soluble epoxide hydrolase inhibitors.
The search for good sEH inhibitors has been actively pursued for the last about twenty years as reviewed by Hammock et al., Comprehensive Toxicology, (Guengerich, F. P., ed.), Pergamon, Oxford, Vol. 3, Chapter 18, pp. 283-305 (1997). Numerous reagents which selectively modify thiols, imidazoles, and carboxyls irreversibly inhibit sEH. Mullin and Hammock, Arch. Biochem. Biophys., 216, pp. 423-429 (1982) disclosed that chalcone oxides were potent inhibitors of sEH, and Dietze et al., Comp. Biochem. Physiolo., 104B, No.2, pp.309-314 (1993) disclosed that trans-3-phenylglycidols were potent chiral inhibitors of sEH.
Copending U.S. Ser. No. 08/909,523, filed Aug. 12, 1997 now U.S. Pat. No. 5,955,496, Hammock et al., suggests the treatment of pulmonary diseases with epoxide hydrolase inhibitors such as chalcone oxides, and describes assays for epoxide hydrolase inhibitors. Among the epoxide hydrolase inhibitors taught are alternative enzyme substrates, such as the epoxide of methyl oleate and other fatty acids and esters or methyl epoxyoctadecenoate and phenyl glycidiols, as well as chalcone oxides.
EH enzymes have been reported to be present in a wide variety of species including bacteria, yeast, fungi, plants, nematodes, insects, birds, fish, and mammals. Indeed, they appear to be present in most, if not all, organisms, and have multiple roles. Plant epoxide hydrolases are also known. For example, fatty acid epoxide hydrolases from apple fruit skin, soybean seedlings, and rice plants have been described. The cDNAs encoding epoxide hydrolase from potato, cress, and tobacco have been isolated and cloned. Stapleton et al., Plant J., 6, pp. 251-258 (1994); Kiyosue et al., Plant J., 6, pp. 259-269 (1994); Guo et al., Plant J., 15, pp. 647-656 (1998). These plant epoxide hydrolases show a high homology with mammalian soluble epoxide hydrolase, but they are 30% shorter on the N-terminus.
Epoxide hydrolases in insects and other arthropods function in the metabolism of endogenous chemical mediators like juvenile hormone and degradation of plant allelochemicals which defend the plant against insects. These enzymes in plants catalyze the hydration of epoxystearic acid to the corresponding .alpha.,.beta.-diols which are important intermediates in the cutin biosynthesis and which have some anti-fungal activity.
Epoxides and diols are key synthetic intermediates in the production of both bulk and speciality organic chemicals. Thus, biosynthetic mechanisms to convert epoxides to diols under gentle, regio and stereospecific conditions are very important. The ability to test if a biosynthetic pathway involves an epoxide by the use of a selective inhibitor can be important in the search for new biosynthetic enzymes and the use of high affinity binding agents in the rapid affinity purification of epoxide hydrolases has proven very important in the study of the mammalian soluble epoxide hydrolases.
The presently known high affinity binding agents for affinity purification of the mammalian soluble epoxide hydrolases include thiols such as benzylthiol, alkyl or terpenoid thiols reacted with epoxy activated SEPHAROSE separation media (SEPHAROSE is available from Pharmacia). These affinity chromatography columns bind a variety of proteins, many of which have a lipophilic catalytic site. The soluble epoxide hydrolase can be selectively eluted from these columns with chalcone oxides, generally as described by Prestwich, Proc. Natl. Acad. Sci. USA, 82, pp. 1663-1667 (1985) and Wixtrom et al., Analyt. Biochem, 169, pp. 71-80 (1988). However, to date there are no affinity purification systems for other epoxide hydrolases which could be used for the initial isolation and cloning of the enzymes, as well as for the isolation of epoxide hydrolases for industrial purposes. New high affinity binding agents for various epoxide hydrolases, particularly for mammalian soluble epoxide hydrolases, remain a useful goal. Also, eluting agents which are competitive, rather then irreversible inhibitors, could be valuable.